Study On The Function Of Host Proteins Vimentin、Rac1 And Galectin-1 During Infection Of Eimeria Tenella

Chicken coccidiosis is a parasitic disease caused by Eimeria of Apicomplexa,which has caused great economic losses to poultry breeding industry.As an obligate intracellular parasite,Eimeria must rely on host cells to complete their complete life cycle.Therefore,it is particularly important to identify the key proteins in the invasion process and explore the molecular mechanism of Eimeria infection for the prevention and control of coccidiosis.Studies have shown that host proteins play an important role in Eimeria infection,but no key host proteins related to Eimeria invasion have been found.In the present studies,the phosphorylated proteomics before and after infected with Eimeria tenella sporozoites in DF-1 cells were analyzed by TMT Mass Tag labeling quantitative phosphorylation proteomics technique.One of the significantly regulated phosphoproteins,Vimentin,and two host proteins,Ras related C3 botulinum toxin substrate 1 and Galectin-1,known to be important to pathogen infection,was selected to further study their role during Eimeria infection.The results lay the foundation for clarifying the relationship between the Eimeria and the host.1.Phosphorylated proteomic analysis of DF-1 cells infected with Eimeria tenellaPhosphorylated proteins play a crucial role in pathogenic infection,but have not been reported in Eimeria infection.In this study,TMT technology was used to analyze phosphorylated proteomics of DF-1 cells before and after E.tenella sporozoites infection.The results showed that a total of 10122 phosphopeptides matched to 3398 host cell phosphoproteins were identified and 13437 phosphorylation sites(including 11396 Serine;1943 Threonine;98 Tyrosine).The comparative analysis of phosphorylated peptides in the uninfected group(UI),post-infection 6 h group(PI6)and post-infection36 h group(PI36)(a fold change > 1.5 or <0.67 and P value<0.05)showed that,PI6/UI group had 731up-regulated phosphorylated peptides and 2 down-regulated;PI36/UI group had 2,189 up-regulated phosphorylated peptides and 27 down-regulated;PI36/PI6 group had 185 up-regulated phosphorylated peptides and 186 down-regulated.Bioinformatics analysis showed that the significantly regulated phosphoproteins(SRPs)in different stages(especially intracellular proliferation)after sporozoites infection were involved in the regulation of cell metabolism.It is suggested that both sporozoites invasion and intracellular development are involved in the regulation of host energy metabolism.It is also found that the regulation of host cytoskeleton by sporozoites persists throughout the infection process.Our study enriches phosphorylated proteomics data of E.tenella infected host cells and provides a new perspective for subsequent related studies.2.Effect of host Vimentin on Eimeria tenella sporozoites invasion of DF-1 cellsVimentin gene was cloned using DF-1 cell cDNA as a template and expressed.q PCR,Western blot and indirect immunofluorescence were used to detect m RNA transcription level,translation level,phosphorylation level and distribution change of Vimentin before and after sporozoites infection respectively.The effect of Vimentin polyclonal antibody or acrylamide incubating DF-1 cells and si RNA down regulating Vimentin expression level on sporozoites invasion were detected by in vitro invasion test.The results showed that the Vimentin gene was successfully cloned and Vimentin-GST recombinant protein was obtained.q PCR and Western blot results showed that Vimentin transcription and translation levels increase continually at 6～72 h after sporozoites infection,and Vimentin phosphorylation levels also changed.The results showed that after infection with sporozoites for 24 h,Vimentin wrapped around the sporozoites to form a cage-like structure.The results of in vitro invasion assay showed that the invasion efficiency of sporozoites was significantly improved by both incubating Vimentin polyclonal antibodies and down-regulating of Vimentin expression in DF-1 cells.However,treatment of DF-1 cells with acrylamide had no significant effect on the invasion efficiency of sporozoites.These results suggest that Vimentin play an inhibitory role during the invasion of sporozoites.3.Effect of host Ras-related C3 Botulinum Toxin Substrate 1 on Eimeria tenella sporozoites invasion of DF-1 cellsRac1 gene was cloned using DF-1 cell cDNA as a template and expressed.q PCR,Western blot and indirect immunofluorescence were used to detect m RNA transcription level,translation level and distribution change of Rac1 before and after sporozoites infection respectively.The effect of Rac1 polyclonal antibody and its inhibitor NSC23766 treated DF-1 cells on sporozoites invasion were detected by in vitro invasion assay.The results showed that the Rac1 gene was successfully cloned and Rac1-GST recombinant protein was obtained.q PCR and Western blot results showed that Rac1 transcription levels were significantly increased at 60 h and 72 h post-infection by sporozoites,while the translation level was not significantly different.The results of indirect immunofluorescence showed that the fluorescence intensity of the Rac1 increased at the top of the sporozoites when the sporozoites invaded the DF-1 cells 15～30 min.The results of in vitro invasion assay showed that DF-1 cells incubated by Rac1 polyclonal antibodies have little effect on the invasion ability of sporozoites,but inhibition of Rac1 endogenous activity with NSC23766 could significantly reduce the invasion efficiency of sporozoites,which is proportional to the concentration of inhibitors.The above results suggest that the endogenous activity of host Rac1 could affects the efficiency of sporozoites invasion of DF-1 cells and Rac1 plays a positive role in the invasion of sporozoites.4.Effect of host Galectin-1 on Eimeria tenella sporozoites invasion of DF-1 cellsGalectin-1 gene was cloned using DF-1 cell cDNA as a template and expressed.q PCR,Western blot and indirect immunofluorescence were used to detect m RNA transcription level,expression and distribution change of Galectin-1 before and after sporozoites infection respectively.The effect of Galectin-1 polyclonal antibody treated DF-1 cells on sporozoites invasion was detected by in vitro invasion assay.The results showed that the Galectin-1 gene was successfully cloned and Galectin-1-GST recombinant protein was obtained.q PCR and Western blot results showed that the transcription and translation levels of Galectin-1 changed dynamically after sporozoites infection.The results of indirect immunofluorescence showed that there was no significant change in the distribution of Galectin-1 after infection by sporozoites.The results of in vitro invasion assay showed that DF-1cells incubated by Galectin-1 polyclonal antibodies have little effect on the invasion ability of sporozoites.Galectin-1 specific role in the invasion of sporozoites needs further study.